Hindbrain Neuroepithelium Research Articles

Serotonin and morphogenesis. Transient expression of serotonin uptake and binding protein during craniofacial morphogenesis in the mouse.

This study describes the timecourse of expression of low-affinity serotonin uptake sites in the developing craniofacial region of the mouse embryo. Whole mouse embryos were incubated in the presence of various serotonergic compounds followed by immunocytochemical localization of serotonin (5-HT) and its binding protein. In the gestational day 9 embryo (3-5 somites), 5-HT uptake was observed in the myocardium of the heart, the visceral yolk sac and foregut. A specific and transient pattern of 5-HT uptake was observed in the hindbrain neuroepithelium from day 9.5-11, where it was localized in rhombomeres 2-5 in the day 9.5 embryo. By day 10, when rhombomeres were no longer evident, uptake was present in the dorso-lateral neuroepithelium surrounding the fourth ventricle (rhombic lip; cerebellar anlage). Uptake of 5-HT was initially observed in the surface epithelium of the craniofacial region at day 10 (20-25 somites) and was greatly increased at day 11. The invaginating lens, nasal placode epithelium and otocyst also took up 5-HT at day 11. During these stages a 45 kD serotonin-binding protein (SBP) was expressed in craniofacial mesenchyme, and became progressively restricted to regions subjacent to epithelial uptake sites. These staining patterns were shown to be specific for 5-HT and SBP by their absence in embryos stained using preabsorbed antisera. The timecourse of these patterns are correlated with critical events in craniofacial morphogenesis including (1) onset of inductive epithelial-mesenchymal interactions, (2) invagination and fusion of placodal structures, (3) presence of rhombomeres, and (4) regions of low proliferative activity.

Neuroectodermal autonomy of Hox-2.9 expression revealed by rhombomere transpositions

Involvement of the Hox genes in regional specifications of the vertebrate body axis is suggested by sequence similarity with the homeotic selector genes of Drosophila, the conservation of a collinear relationship between genomic organization and site of expression, and mutational analysis. Subdivision of vertebrate embryo hindbrain neuroepithelium into lineage compartments (rhombomeres) underlies segmental patterning of neuronal differentiation. The rhombomere boundaries delimit domains of expression of Hox genes, presumed to be determinants of rhombomere phenotype, suggesting that Hox genes confer positional value; the formation of rhombomere 4 (r4) is followed by strong expression of Hox-2.9 within its confines. If the Hox genes are determinants, their expression should be autonomous from the developmental stage at which regional commitment becomes fixed and irreversible. We have transplanted the future r4 region (from state-9-chick embryos) into the more anterior position of r2 and probed for Hox-2.9 transcripts. We report here that Hox-2.9 was expressed in the ectopic r4 as strongly as in the normal r4, whereas reciprocal grafts of future r2 to r4 position did not express Hox-2.9. The phenotype of ectopic rhombomeres developed according to their original position, as demonstrated by retrograde tracing of efferent cranial nerve nuclei. As early as stage-9-(six somites), both Hox-2.9 expression and segment identity are autonomous in the chick embryo hindbrain, independent both of position in the neuroepithelium and of signals from the underlying mesoderm.

Teratogenic effects on the neuroepithelium of the CD‐1 mouse embryo exposed in utero to sodium valproate

A causal association has now been recognized between the use of the anticonvulsant drug sodium valproate during pregnancy and the increased incidence of spina bifida in the human population. The objective of this study was to investigate the teratogenic effects of sodium valproate on the cephalic 1) neuroepithelium, 2) extracellular matrix, and 3) embryonic protein content in the CD-1 mouse embryo. Nulliparous female CD-1 mice were dosed intraperitoneally on day 8 of gestation with 340 mg/kg of sodium valproate. On day 10 of gestation, females were killed by cervical dislocation, and all live embryos were assigned to one of the following groups and processed accordingly for: 1) head measurements, 2) scanning electron microscopy, 3) total protein determination, 4) two-dimensional polyacrylamide gel electrophoresis, 5) immunohistochemistry, and 6) light microscopy. Exposure to sodium valproate at the selected dosage resulted in a 30% incidence of neural tube defects in the cranial region of these embryos. Treated embryos showed a significant reduction in head size, indicating a drug-induced microcephaly. No major differences were seen in the total embryonic protein patterns between control and treated embryos. Immunoreactivity to laminin and fibronectin showed a similar distribution in control and treated embryos except in the vasculature pattern of the hindbrain neuroepithelium. The neuroepithelium of the treated embryos showed marked disorganization when it was examined histologically, particularly in the forebrain region. Cells were disoriented, and there was a noticeable loss of intercellular adhesion in the juxtaluminal region. Increased cellular blebbing was apparent at the ependymal surface, and large protrusions of cells were seen invading the neural tube lumen. The lumen was distorted in shape and frequently contained blood cells. Irregularities and gaps were observed in the underlying basal lamina. These results suggest that treatment with sodium valproate during a critical time in neurogenesis in the CD-1 mouse embryo alters the normal architecture of the neuroepithelium, with a loss of integrity at both the basal and apical surfaces. The alterations seen in the neuroepithelium at any of these sites in this animal model could help explain the increased incidence of spina bifida seen in children of epileptic mothers receiving sodium valproate.

Fluidity of the neural epithelium during forebrain formation in rat embryos.

During neurulation in rat embryos, the forebrain grows more rapidly than can be accounted for by intrinsic cell division alone, while the adjacent midbrain/rostral hindbrain maintains a constant cell number despite a high mitotic index (the cell cycle time is 6 h throughout the neural epithelium). We have proposed that neuroepithelial cells flow in a rostral direction within the midbrain/rostral hindbrain region, towards and into the rapidly expanding forebrain. Evidence in support of this hypothesis is provided by cell-labelling studies: labelled neuroepithelial cells were injected into specific sites in the midbrain or rostral hindbrain neuroepithelium of unlabelled embryos; after culture of these embryos for 24 h, the labelled cells were found in positions rostral to the injection sites. In the midbrain/rostral hindbrain region, mitotic spindles were found to be predominantly orientated parallel with the long axis of the embryo; transversely orientated spindles were more frequently observed close to the lateral edges than more medially. Neural crest cells emigrate from the lateral edges during neurulation. These observations suggest that mitotic spindle orientation reflects the direction of cell movement: in the lateral region movement towards the lateral edge would maintain cell number in the transverse plane as neural crest cells emigrate; elsewhere, cells are moving mainly in the longitudinal plane, towards the forebrain. The possible causal relationship between cell movement and mitotic spindle orientation is discussed. Cell flow within the intact neural epithelium is compared with cell flow in the intestinal villus epithelium. Other types of epithelial cell movement observed in the cranial neural epithelium during neurulation include expansion and shrinkage of the epithelial surface associated with change of cell shape and microfilament-mediated curvature. Cell rearrangement involving exchange of neighbours and cell movement towards and into a site of epithelio-mesenchymal conversion are also implicated.